Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Affymetrix SNP array data for hepatocellular carcinoma tissue

ABSTRACT: Genetic variations play an important role in tumor development and metastasis. Hepatocellular carcinoma (HCC) is one of leading cause of cancer-related death. Despite improvements in surveillance and clinical treatment strategies, the prognosis of HCC remains dismal. Affymetrix SNP 6.0 array were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent liver transplantation. Recurrence related SNPs were selected and validated. Affymetrix SNP 6.0 arrays were performed according to the manufacturer's directions on DNA extracted from formalin-fixed paraffin-embedded HCC tissues

ORGANISM(S): Homo sapiens

SUBMITTER: jie hu

PROVIDER: E-GEOD-29667 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genetic variations in plasma circulating DNA of HBV-related hepatocellular carcinoma patients predict recurrence after liver transplantation.

Hu Jie J Wang Zheng Z Fan Jia J Dai Zhi Z He Yi-Feng YF Qiu Shuang-Jian SJ Huang Xiao-Wu XW Sun Jian J Xiao Yong-Sheng YS Song Kang K Shi Ying-Hong YH Sun Qi-Man QM Yang Xin-Rong XR Shi Guo-Ming GM Yu Lei L Yang Guo-Huan GH Ding Zhen-Bin ZB Gao Qiang Q Tang Zhao-You ZY Zhou Jian J

PloS one 20111005 10

<h4>Background</h4>Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis.<h4>Methods</h4>Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphis ...[more]

PMID: 21998744

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Project description:In the study of tumor genetics, formalin-fixed paraffin-embedded (FFPE) tumors are the most readily available tissue samples. While DNA derived from FFPE tissue has been validated for array comparative genomic hybridization (aCGH) application, the suitability of such fragmented DNA for single-nucleotide polymorphism (SNP) array analysis has not been well examined. Furthermore, whole-genome amplification (WGA) has been used in the study of small precursor lesions to produce sufficient amount of DNA for aCGH analysis. It is unclear whether the same approach can be extended to SNP analysis. In this study, we examined the utility and limitations of genotyping platform performed on whole-genome amplified DNA from FFPE tumor samples for both copy number and SNP analyses. We analyzed the results obtained using DNA derived from matched FFPE and frozen tissue samples on Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol), were used to determine how WGA methods affect the results. We found that the use of DNA derived from FFPE tumors (without or with WGA) for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large-scale chromosome LOH (>10Mb) can be reliably detected from FFPE tumor DNA samples (without or with WGA) but not smaller LOH or copy number alterations. Our findings here indicate a need for caution in SNP array data interpretation when using FFPE tumor-derived DNA, particularly with WGA. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved and FFPE mesenchymal tumor samples without or with WGA, as well as genomic snap-frozen non-neoplastic tissue DNA from 5 adult individuals to serve as reference DNA. WGA was performed using the REPLI-g® FFPE kit (Qiagen, Valencia, CA, USA) and GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA) in parallel in accordance with the manufacturers’ protocols. A two- to eight-hour individualized reaction time was used in the Qiagen platform for each sample. A gradient amount of initial DNA (10ng, 30ng, 60ng, 100ng and 150ng) was tested followed by gel electrophoresis and qualitative multiplex PCR assay to determine the quality of post-WGA products. At least four independent experiments were concurrently performed per template amplification. Four separateWGA reaction products were pooled for each sample for subsequent microarray analysis to minimize the amplification bias and allele dropout. One of the Affymetrix GeneChip® Human Mapping 500K Array Set (Nsp 250K SNP array) was used for genotyping analysis. Four gastrointestinal stromal tumors with known cytogenetic aberrations were included. Two cases were sucessfullly amplified and passed the quality tests. A total of 12 samples were compared between each other, including frozen tissue DNA (as reference), frozen tissue DNA with WGA (two platforms), FFPE tissue DNA, and FFPE tissue DNA with WGA (two platforms) from each case.

Project description:Methods for haplotyping and DNA copy number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required that substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP-genotypes (AA, AB, BB), and DNA copy number profiling remains difficult as true DNA copy number aberrations have to be discriminated from WGA-artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by deciphering WGA-distorted SNP B-allele fractions, using a process we coin haplarithmisis. We demonstrate clinical precision of the method on single cells biopsied from human embryos to diagnose disease alleles genome wide, we advance and facilitate the detection of numerical and structural chromosomal anomalies in single cells, and can distinguish meiotic from mitotic segregation errors in a single assay. The samples of a reference family were applied for optimisation of single-cell genotyping using Affymetrix SNP-arrays prior to downstream analysis. Specifically, the reference family delivers genomic DNA samples isolated from peripheral blood of two siblings 'S1' and 'S2', the mother and father of these siblings, as well as of the maternal grandmother and grandfather. Of individuals ‘S1’ and ‘S2’, six EBV-transformed lymphoblastoid single cells were isolated of which three were whole-genome amplified using MDA and three using PicoPlex. These WGA-products were hybridized to Affymetrix NspI 250K SNP-arrays following the protocol as recommended by the company. Subsequently, the SNP-probe signals were interpreted by different genotyping algorithms (see data processing). Based on overall performance, it was decided to use the Dynamic Model (DM) for interpreting Affymetrix SNP-probe signals of single cells.

Dataset Information

Affymetrix SNP array data for hepatocellular carcinoma tissue

Publications

Genetic variations in plasma circulating DNA of HBV-related hepatocellular carcinoma patients predict recurrence after liver transplantation.

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